Phospholipids.
Phospholipids are a class of amphipathic phosphorus containing lipids which are essential constituents of biological membranes. Various phospholipid preparations have been used for cooking, drug delivery (liposomes), slow release delivery systems, carrier media for hydrophobic drugs, gene transfer and replacement therapy, sunscreens, emulsions, anti-foaming agents, replacement of damaged or absent pulmonary surfactants, detergents and membrane stabilization. Phosphatidic acid (PA), phosphatidylinositol (PI), lysophosphatidic acid, lysophosphatidylinositol (LPI), and lysophosphatidylcholine (LPC) are found in a variety of plant and animal products. Lysophosphatidic acid analogs have been reported to have a variety of physiological activities including mitogenesis (i.e. prevention of hyperproliferative diseases), vasodilation, growth factor, wound healing and to be an anti-wrinkle agent. In addition, previous studies have shown that lysophosphatidic acid, when bound to serum albumin, can activate membrane currents in Xenopus oocytes and induce neurite retraction in PC12 pheochromocytoma cells.
Apoptosis.
Cell death can occur by necrosis and apoptosis is generally results from catastrophic irreversible cell damage. It is characterized by early swelling of the cell and its cytoplasmic organelles, with subsequent rupture of the cellular membrane.
Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Apoptotic cell death appears to play a significant role in the tissue damage that occurs in association with, for example, ischemia, organ transplantation, and various gastrointestinal disorders.
Studies of apoptosis suggest that a common metabolic pathway leading to apoptosis can be initiated by a wide variety of signals, including hormones, serum growth actor deprivation, chemotherapeutic agents, ionizing radiation, and infection by human immunodeficiency virus (HIV). Apoptosis can also be induced by mild, non-catastrophic cell injury and can be concomitant with adjacent necrosis. Agents that affect the biological control of apoptosis thus have therapeutic utility in numerous clinical indications.
Apoptotic cell death is characterized by morphologic changes such as cellular shrink age, chromatin condensation and margination, cytoplasmic blebbing, and increased membrane permeability. Specific internucleosomal DNA fragmentation is a hallmark for many, but not all, instances of apoptosis.
Several genes and gene families involved in signal transduction and modulation of apoptosis have been described. Apoptosis, however, is an active cellular response to a physiologic or external signal and can be modulated by interfering with the apoptotic pathway. Conversely, by definition, necrosis can be prevented only by decreasing cell injury. Prevention of apoptosis by upregulation of bcl-2 and bcl-x expression, or by inhibitors of ICE-like proteases are typical examples of modulation of cell death. Umansky Molekylyarnaya Biologiya 30:285-295 (1996); Vaux and Straser PNAS 93:2239-2244 (1996); Nunez et al. (1994) Immunol. Today 15:582-588; and Whyte (1996) Trends in Cell Biol. 6:245-148.
Ischemia and Reperfusion.
Ischemia is the result of decreased blood flow to a particular area or organ of the body. Ischemia is responsible for several important types of physiologic damage such as brain damage, spinal cord trauma and myocardial ischemia. Acute myocardial ischemia leads to the death of individual heart cells which can result in organ dysfunction. Although early reperfusion (i.e., restoration of blood flow) decrease heart damage caused by ischemia, cell death by apoptosis can occur upon reperfusion. In this instance, the cells that die are those that remained viable at the end of ischemia. Support for the role of apoptosis in heart injury induced by ischemia and subsequent perfusion has been provided by numerous laboratories. Gottlieb et al. (1994) J. Clin. Invest. 94:16211628; Umansky et al. (1995) Cell Death and Differentiation 2:235241; Umansky et al. (1996) Basic and Applied Myology 6:227235; and Itoh et al. (1995) Am. J. Pathol. 146:13251331. Severe cell damage during prolonged ischemia appears to result in necrotic death of myocardial cells. However, if the ischemia is relatively limited in extent and duration, the apoptotic pathway is initiated. Restoration of blood flow (reperfusion) allows apoptosis to proceed. Insulin-like growth factors (IGF) and calpain inhibitors, which are capable of preventing apoptosis in different systems, also inhibited apoptosis of cardiomyocytes following ischemia and reperfusion both in vivo and in vitro. Umansky et al. (1995); and Buerke et al. (1995) Proc. Natl. Acad. Sci. USA 92:80318035.
Organ Preservation.
Transplantation of vital organs such as the heart, liver, kidney, pancreas, and lung has become increasingly successful and sophisticated in recent years. Because mammalian organs progressively lose their ability to function during storage, even at freezing temperatures, transplant operations need to be performed soon after obtaining a donor organ to minimize the period of time that the organ is without blood flow. This need diminishes the availability of organs for transplantation.
In clinical practice, the two major situations in which cardiac preservation is required are heart transplantation and cardioplegia for open heart surgery. In heart transplantation, the donor heart is flushed with approximately 1 liter of cold organ preservation solution (OPS) to arrest the heart. Cooling is supplemented by surrounding the heart with iced saline. The chilled, arrested heart is then surgically excised, immersed in cold OPS, packed in ice and rushed to the recipient center.
After placing the recipient on cardiopulmonary bypass, the diseased heart is excised. The preserved donor heart is then removed from the OPS, trimmed appropriately and transplanted into the recipient. Blood is allowed to flow to the transplanted heart. The transplanted heart will then either resume beating spontaneously or will require chemical and/or electrical treatment to restore normal rhythm. When the heart is ready to take over the circulation, the cardiopulmonary bypass is discontinued and the recipient's chest closed.
Most non-transplant surgical procedures on the heart, such as coronary artery bypass grafting, require that the heart be arrested for a period ranging from 1 to 4 hours. During this time, the heart is kept cool by external cooling as well as by periodically reflushing an OPS through the coronary arteries. The OPS composition is designed to rapidly arrest the heart and to keep it in good condition during the period of standstill so that it will resume normal function when the procedure is finished.
In cardioplegic procedures, the heart is exposed in the chest and, at a minimum, the aortic root is isolated. A vascular clamp is applied across the aorta and approximately 1 liter of cold OPS is flushed into the aortic root through a needle. Venting is provided through the left ventricle, pulmonary artery or the right atrium and the effluent, which can contain high levels of potassium, is suctioned out of the chest. This, together with external cooling, produces rapid cessation of contractions. During the period of arrest, the patient's circulation is maintained artificially using cardiopulmonary bypass.
After completion of the surgical procedure, blood flow is restored to the coronary circulation and heartbeat returns either spontaneously or after chemical and electric treatment. The ease with which stable function is restored depends to a large extent on the effectiveness of the OPS to preserve the heart. Once the heart is beating satisfactorily, cardiopulmonary bypass is discontinued and the chest closed.
It is generally understood that “living” organs, including the heart, continue the process of metabolism after removal from the donor so that cell constituents are continuously metabolized to waste products. If the storage technique is inadequate, the accumulation of these metabolic waste products, depletion of cell nutrients and consequent derangement of cell composition lead to progressive loss of function and ultimately to cell death, with loss of adequate function after transplantation into the recipient. Several procedures have been explored to successfully enable organ preservation ex vivo for useful time periods. In one method, the donor organ is cooled rapidly by flushing cold solutions through the organ's vascular system and maintaining the organ at temperatures near 0° C. for the purpose of greatly slowing the metabolic rate. In the case of the mammalian heart, the flush solution composition is designed to cause the heart to rapidly stop beating as well as to preserve it.
In 1988, University of Wisconsin (UW) solution was introduced. Belzer et al. (1988) Transplantation 45:673-676. This solution, capable of preserving the pancreas and kidney for 72 hours, and the liver for 30 hours, subsequently became the standard organ preservation solution (OPS) for transplant surgery and the benchmark against which other OPS compositions were measured. However, the heart is more recalcitrant to long-term storage than other organs, and UW solution is unreliable for storage of hearts for as short a period as 24 hours. Wicomb et al. (1989) Transplantation 47:733-734.
Improvements in the design of OPS compositions, as reviewed in Collins et al. (1992) Kidney International 41:S-197-S-202 and others described in the art, have proceeded along several paths, including: (1) modification and simplification of UW solution; (2) investigation of organ-specific requirements; (3) addition of pharmacologic agents, particularly calcium antagonists for control of acidosis; (5) the use of a terminal rinse solution; and (6) the use of solutions containing PEG.
Wicomb et al. reported the beneficial effects of a solution of PEG 8000 and horseradish peroxidase on rabbit hearts preserved by oxygenated low pressure perfusion for 24 hours. Wicomb et al. (1989) Transplantation Proceedings 21:1366-1368. The substitution of PEG20M as the colloid for hydroxyethyl starch (HES) of the UW solution also yielded excellent cardiac function. PEG20M consists of two or more molecules of PEG 6000-8000 joined by a bisphenol epoxide linker (CAS #37225-26-6; CAS name Oxirane, 2,2′[(1-methyl-ethylidene)bis(4,1-phenyleneoxy methylene)]bis-, polymer with (-hydro-(-hydroxypoly(oxy-1,2-ethanediyl). The substitution of PEG20M for HES also allowed baboon heart storage up to 48 hours and increased cardiac output (CO) under conditions of microperfusion. Wicomb et al. (1986) J. Surg. Res. 40:276; and Wicomb et al. (1989) Transplantation 48:6-9. “Microperfusion” is a hypoxic, very-low-flow perfusion with flowrates such as 3 ml/g heart wt/24 hour, which is 1/500 of that typical of conventional continuous perfusion. Wicomb et al. (1989) Transplantation 48:6-9.
An improved OPS, Cardiosol™ heart preservation solution, contained the substitution of PEG20M for HES and eliminated five components of UW solution (penicillin, dexamethasone, insulin, allopurinol, and adenosine). Wicomb et al. (1990) Transplantation 49:261-264; and U.S. Pat. No. 4,938,961. Cardiosol™ heart preservation solution contains 5% or 10% by weight PEG 20M (Union Carbide Chemicals and Plastics Co., Inc., Charleston, W. Va.), 40 mM sodium, 125 mM potassium, 5 mM magnesium, 25 mM phosphate, 5 mM sulfate, 100 mM lactobionate, 30 mM raffinose, and 3 mM glutathione. Collins et al., The Lancet 338:890-891 (1991); and Wicomb et al. (1994) J. Heart Lung Transplantation 13:891-894. This solution was found to be superior to UW solution both for 4-hour hypothermic and 24-hour microperfusion storage. Collins et al. (1992).
Gastrointestinal Disorders.
A variety of food supplements containing, in part, partially processed plant extracts have been used to ameliorate the gastrointestinal disorders that often accompany chemotherapy, radiation and AIDS. The supplements generally contain carbohydrates, fat and plant protein hydrolysates. See, e.g., Tomei and Cope et al. in Apoptosis: The Molecular Basis of Cell Death (1991) Cold Spring Harbor Laboratory Press. PCT Publication No. WO 95/15173, U.S. Pat. Nos. 5,620,885, 5,567,425, 5,635,186 and 5,624,672 describe plant-derived extracts that produce an anti-apoptotic effect. It has now been found that these extracts contain the following phospholipids: LPA, LPC, LPI, PA and PI in the ratios of approximately 2:1:2:20:20, by weight in addition to various optional protein and carbohydrate constituents.
A need exists for improved solutions and methods for preserving or restoring cell, tissue, or organ function, and/or preventing apoptosis for a variety of therapeutic uses, particularly organ preservation. The present invention satisfies this need and provides related advantages, as well.